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Structural, biosynthetic and serological cross-reactive elucidation of capsularpolysaccharides from Streptococcus pneumoniae serogroup 16
Li, Chengxin; Duda, Katarzyna A.; Elverdal, Pernille L.; Skovsted, Ian C.; Kjeldsen, Christian; Duus, JensØ.
Published in:Journal of Bacteriology
Link to article, DOI:10.1128/JB.00453-19
Publication date:2019
Document VersionPeer reviewed version
Link back to DTU Orbit
Citation (APA):Li, C., Duda, K. A., Elverdal, P. L., Skovsted, I. C., Kjeldsen, C., & Duus, J. Ø. (2019). Structural, biosyntheticand serological cross-reactive elucidation of capsular polysaccharides from Streptococcus pneumoniaeserogroup 16. Journal of Bacteriology, 201(20), [e00453-19]. https://doi.org/10.1128/JB.00453-19
1
Structural, biosynthetic and serological cross-reactive elucidation of 1
capsular polysaccharides from Streptococcus pneumoniae serogroup 16 2
Chengxin Lia, Katarzyna A. Dudab, Pernille L. Elverdalc, Ian C. Skovstedc, Christian Kjeldsena, 3
Jens Ø. Duusa,* 4
a Department of Chemistry, Technical University of Denmark, Kgs. Lyngby, Denmark 5
b Junior Research Group of Allergobiochemistry, Research Center Borstel, Leibniz Lungenzentrum, 6
Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, 7
Germany 8
c SSI Diagnostica A/S, Hilleroed, Denmark 9
10
*Corresponding author: jduus@kemi.dtu.dk, +45 45252451 11
Abstract 12
Capsular polysaccharides (CPS) are crucial virulence factors of Streptococcus pneumonia. The 13
previously unknown CPS structures of the pneumococcal serogroup 16 (serotype 16F and 16A) 14
were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by 15
chemical analysis. The following repeat unit structures were determined: 16
17 A further analysis of CPS biosynthesis of serotype 16F and 16A, in conjunction with published cps 18
gene bioinformatics analysis and structures of related serotypes, revealed presumable specific 19
function of glycosyltransferase, acetyl transferase, phosphotransferase and polymerase. The 20
JB Accepted Manuscript Posted Online 5 August 2019J. Bacteriol. doi:10.1128/JB.00453-19Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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functions of glycosyltransferase WcxN and WcxT were proposed for the first time, which were 21
assigned to catalyze linkage of α-L-Rhap-(1-3)-α-D-Glcp and α-L-Rhap-(1-2)-α-L-Rhap, 22
respectively. Furthermore, since serotype 16F was genetically close to serogroup 28, cross-reactions 23
between serogroup 16 and serogroup 28 were studied using diagnostic antisera, which provided 24
further understanding of antigenic properties of CPS and diagnostic antisera. Interestingly, serotype 25
16F cross-reacted with factor antisera 28b and 11c. Meanwhile, serotype 16A cross-reacted with 26
factor antiserum 11c. 27
Importance: 28
The vaccine pressure against Streptococcus pneumoniae could result in the change of prevalence in 29
carriage and invasive serotypes. As such, it is necessary to monitor the distribution to achieve 30
successful vaccination of the population, and similarly, it is important to increase the knowledge of 31
even the currently less prevalent serotypes. The CPS are vital for the virulence of the pathogen and 32
antigenic properties of CPS are based on the structure. Consequently, a better understanding of the 33
structure, biosynthesis and serology of the capsular polysaccharides can be of great importance 34
towards developing future diagnostic tools and vaccines. 35
INTRODUCTION 36
Streptococcus pneumoniae is an important encapsulated Gram-positive human pathogen, with 93 37
distinct serotypes reported (1)(2). The capsular polysaccharide (CPS), forming the hydrated outer 38
layer of S. pneumoniae, protects the pathogen from the host immune system, which is the basis for 39
successful vaccines against infections caused by S. pneumoniae. Based on the importance of CPS, it 40
has been extensively studied from basic structure and genetic biosynthesis to immunology. 41
Determination of unknown pneumococcal CPS structures is important to understand the function of 42
biosynthetic genes, enzymes and relationships between structure and antigenicity. The elucidation 43
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of genetic basis for all capsular types have been completed by sequencing the corresponding DNA 44
stretches (3). While, the structural analysis of the corresponding capsular polysaccharide has 45
remained incomplete. 46
Serotype 16F is not included in any current vaccine, however, in recent years, it was reported as a 47
potential cause of Invasive Pneumococcal Disease (IPD) and increased case-fatality rates in several 48
studies (4)(5)(6)(7)(8). The efficacy of a given vaccine is inherently dependent on the serotype 49
distribution in the target population for the serotype-specific protection of pneumococcal vaccines. 50
Thus, it is important to monitor the epidemiology of pneumococcal serotypes to formulate future 51
pneumococcal vaccines. One study proposed 16F as a candidate for higher valency pneumococcal 52
conjugate vaccines (9). However, the CPS structure of serotype 16F and 16A were both unknown, 53
which consequently resulted in ambiguity of the function of related biosynthetic genes and enzymes. 54
Here we report the repeat unit structure of serotype 16F and 16A CPS based on NMR and chemical 55
analysis, together with biosynthetic analysis to correlate CPS structure with the presence of related 56
enzymes. Then, assignments of specific functions of glycosyltransferases, acetyltransferase, 57
phosphotransferases and polymerases is proposed. In addition, cross-reactions with rabbit antisera 58
between serogroup 16 with genetically related serotypes were studied to gain understanding of CPS 59
from serological perspectives. 60
RESULTS AND DISCUSSION 61
NMR structural analysis of 16F and 16A CPS repeat units were conducted mainly in two steps: 62
assignment of individual monosaccharides or residues and connecting the identified structural 63
motifs to yield the repeat unit structure (10). 64
Assignment of individual spin systems were based on 1D 1H NMR and 2D double quantum filter 65
correlated spectroscopy (DQF-COSY) spectra, total correlation spectroscopy (TOCSY) spectra, 66
multiplicity edited 1H–13C heteronuclear single quantum coherence (HSQC) spectra and HSQC-67
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TOCSY spectra. In addition, heteronuclear 2 bond correlation (H2BC) spectra and heteronuclear 68
multi-bond correlation (HMBC) spectra were used to confirm spin systems, especially in some 69
overlapping regions. Then glycosidic bonds were identified using 1H–13C HMBC spectra and 70
nuclear Overhauser effect spectroscopy (NOESY) spectra, and similarly were O-acetylated 71
positions. Phosphodiester linkages were determined by 1H–31P HMBC. Clean in-phase (CLIP) -72
HSQC (11) was used to determine the anomeric configuration of pyranosides (12) . In addition, 73
NMR analysis of the de-O-acetylated (de-OAc) CPS and dephosphorylated (de-P-Gro) CPS was 74
conducted to simplify spectra and confirm the position of substitutions. 75
The 16F CPS NMR spectra gave broad lines because of short transverse relaxation times (T2) due 76
to slow molecular tumbling expected from high molecular weight polysaccharides, but could also 77
be partly due to the heterogeneous nature of the sample. Applying higher temperature (333k) and 78
slightly depolymerizing CPS by sonication can increase molecular tumbling rate in order to 79
improve line shapes. Moreover, removal of OAc groups and P-1-Gro substituents also lead to 80
narrower lines as the sample became more homogeneous. There were a relatively strong 81
phosphocholine signal at 3.2/54.5 ppm and other small signals from cell wall polysaccharides 82
(CWPS)(13) in both 16F and 16A CPS NMR spectra. 83
NMR assignments for serotype 16F CPS repeat unit 84
According to 1D 1H and HSQC spectra of 16F CPS, there were four different anomeric signals 85
corresponding to four sugar residues, labelled as A, B, C, and D in order of descending 1H chemical 86
shift (Fig. 1). Furthermore, there also was a downfield signal at 5.634 ppm corresponding to an OAc 87
substitution as well as three methyl signals, two of which corresponded to the 6-position of 6-deoxy 88
sugars as well as one corresponding to an OAc group. Residues A and B were both identified as 89
rhamnopyranosides as they had methyl groups at their 6-position and 3JH, H coupling patterns (Table 90
S1) corresponding to 6-deoxy mannopyranoside configurations. Residue C was determined to be an 91
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α-glucopyranose according to 3JH, H coupling pattern. D was determined to be a β-glucopyranose, as 92
it only showed large 3JH, H coupling constants. The 3JH3, H4 were difficult to measure because of the 93
overlap in both 1H and DQF-COSY spectra. Based on 1JH1, C1 measured from CLIP-HSQC, A and 94
C, with 1JH1, C1 of about 170Hz, were identified as α-configuration, while B and D, with 1JH1, C1 of 95
about 160Hz, were β-configuration(12). The monosaccharide composition analysis showed that 16F 96
CPS were composed of rhamnose and glucose in a 1:1 ratio (Table 1), which was consistent with 97
the results of the NMR analysis. 98
After the monosaccharide units were identified, HMBC and NOESY were used to find the linkage 99
of these monosaccharides (Table 2 and Fig. S1). The anomeric position of A had HMBC and 100
NOESY correlations with C3, while the anomeric position of C had correlations with B3. Similarly, 101
there were HMBC and NOESY cross peaks between B1 and D4 and between D1 and A3. 102
Therefore, these four sugar residues make up the backbone of the repeat unit in the order: 3)-A-(1-103
3)-C-(1-3)-B-(1-4)-D-(1-. Furthermore, according to 31P and 1H-31P HMBC spectra, there were two 104
phosphate groups attached to the backbone. One connected to the 4-position of the α-rhamnose A 105
and another attached to the 6-position of the α-glucose C. The phosphorylated positions were 106
confirmed by NMR analysis of de-P-Gro 16F CPS, as the chemical shifts of A4 (4.110/75.72 ppm) 107
and C6 (4.058/64.70 ppm) in sonicated CPS moved upfield to 3.604/71.38 ppm and 108
3.772/3.779/60.78 ppm in de-P-Gro 16F CPS, respectively. The β-rhamnose B was 2-OAc 109
substituted according to HMBC correlations between carbonyl of the OAc group and B2 proton, 110
which could explain an extremely downfield proton chemical shift of B2 at 5.634 ppm. Moreover, 111
in the NMR spectra of de-OAc 16F CPS, the signals from the acetyl group disappeared and the B2 112
proton signal moved upfield from 5.634 ppm to 4.269 ppm. Furthermore, there were three 113
additional sharp and intense signals, two CH2 groups and one CH group, in sonicated CPS and de-114
P-Gro CPS, which arose from free glycerol. These signals had almost identical chemical shifts and 115
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line shapes to those detected from a reference sample of glycerol in D2O (Fig. S2) and did not show 116
any correlations to other units. The free glycerol could be generated during strong sonication. 117
Therefore, one needs to pay extra attention when using strong sonication to depolymerize large 118
polysaccharides. 119
Based on the above analysis, the repeat unit of serotype 16F CPS was determined to be a 120
tetrasaccharide: -3)-α-L-Rhap-[4-P-1-Gro]-(1-3)-α-D-Glcp-[6-P-1-Gro]-(1-3)-β-L-Rhap-[2-OAc]-(1-121
4)-β-D-Glcp-(1- (Fig.2). Full assignments of the 16F CPS, de-OAc 16F CPS and de-P-Gro 16F CPS 122
repeat unit is given in Table 2. 123
NMR assignments for serotype 16A CPS repeat unit 124
1D 1H and HSQC spectra of 16A CPS contained eight different anomeric signals. However, the 125
intensity of these anomeric signals varied, and it turned out to be due to partial 2-OAc substitution 126
on a single position (Fig. 3). Moreover, NMR analysis of de-O-acetylated 16A CPS showed only 127
six anomeric signals, which indicated that 2-OAc substitution affected nearby anomeric signals. The 128
monosaccharide units were labelled A, B, C, D, E and F in order of descending 1H chemical shift. 129
As A was only partially 2-OAc substituted, A without 2-OAc substitution was labelled as A’. 130
Similarly, the anomeric position of E was affected by the acetylation of A, and as such the 131
corresponding monosaccharide unit in the approximately 30% non-acetylated repeat unit was 132
labelled as E’. 133
Furthermore, three methyl groups were observed, two of which had almost identical chemical shift 134
corresponding to the methyl of 6-deoxy sugars, and the third one was the methyl of an acetyl group. 135
A was identified as a galactose, approximately 70% of which was 2-OAc substituted according to 136
integration of 1H-NMR anomeric signals. The 3JH, H coupling constants of A did not show regular 137
pattern as pyranose sugar, and based on the chemical shifts A was identified as a β-galactofuranose 138
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(14)(15). This 2-OAc substitution of A would explain the downfield shifted proton chemical shift of 139
A2 at 5.155 ppm. The de-OAc 16A CPS spectra became far simpler compared to native 16A CPS, 140
as signals caused by the partial O-acetylation disappeared. The anomeric region of de-OAc 16A 141
CPS showed only six signals with almost identical integrals in the 1H spectrum. The chemical shift 142
of A2 moved upfield from 5.155/82.45 ppm to 4.342/80.45 ppm (Table 3). B and D were both 143
identified as rhamnopyranosides, because of the methyl groups at their 6-positions and the 3JH, H 144
coupling constants that corresponded to 6-deoxy mannopyranose configurations. C and F was 145
identified as an α- and β-galactopyranoside, respectively, according to their 3JH, H coupling 146
constants (Table S1) and chemical shifts. The 6-position of C was connected to a glycerol-1-147
phosphate substitution according to 1H-31P HMBC, which can explain the relatively downfield 148
chemical shift 3.960/64.46 ppm at C6 compared to 3.704/61.24 ppm in de-P-Gro 16A CPS. E was 149
determined to be a β-glucopyranose as it only had large 3JH, H coupling constants. Based on the 1JH1, 150
C1 coupling constants of approximately 170 Hz, measured from CLIP-HSQC, the two rhamnosides 151
B and D were identified as having α-configuration. Additionally, the CLIP-HSQC was also used to 152
confirm the anomeric configurations of C, E and F with 1JH1, C1 coupling constants of 153
approximately 170, 160 and 160Hz, respectively. The anomeric configurations are in agreement 154
with 3JH1, H2 coupling constants and the chemical shifts in general. NMR analysis of the 155
monosaccharide repeat units were in agreement with the results of the monosaccharide composition 156
analysis: rhamnose : galactose : glucose in approximately a 2 : 3 : 1 ratio (Table 1). Furthermore, in 157
the HMBC and NOESY spectra (Fig. S3), cross peaks of A1 with D3, D1 with B2, B1 with C3, C1 158
with F3, F1 with E4 and E1 with A3 revealed linkages of these monosaccharide units. So, these six 159
sugars constitute a linear backbone of the 16A repeat unit in the sequence: 3)-A-(1-3)-D-(1-2)-B-(1-160
3)-C-(1-3)-F-(1-4)-E-(1-. Thus, the repeat unit of serotype 16A CPS were determined to be a 161
hexasaccharide with the following structure: -3)-β-D-Galf-[2-OAc](70%) -(1-3)-α-L-Rhap-(1-2)-α-162
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L-Rhap-(1-3)-α-D-Galp-[(6-P-1)-Gro] (1-3)-β-D-Galp-(1-4)-β-D-Glcp-(1- (Fig.2). The full 163
assignment of the 16A CPS, de-OAc, de-P-Gro 16A CPS repeat unit is given in Table 3. 164
The 13C chemical shift assignments of 16F and 16A CPS also supported glycosidic linkage sites 165
determined by HMBC/NOESY, as all substituted positions had downfield chemical shifts compared 166
to the same unsubstituted position (16). The assigned chemical shifts of de-P-Gro 16F and 16A CPS 167
are in good agreement with those calculated by CASPER(17). 168
Genetic elucidation of serotype 16F and 16A CPS biosynthesis. 169
Biosynthesis of pneumococcal serotype 16F and 16A CPS use the Wzy-dependent mechanism. A 170
summary of 16F and 16A CPS biosynthetic genes, their functions and related serotypes are shown 171
in Table 4, which is based on published genetic sequence data, bioinformatics analysis and CPS 172
structures (3)(18)(19)(1). The cps loci of 16F and 16A did not show high similarity(Fig.4). They 173
have similar conserved regulatory genes cpsABCD (wzg, wzh, wzd and wze), the initial glucosyl-1-174
phosphate transferase gene (wchA)(20) and d-TDP L-rhamnose pathway gene(rmlA, rmlC, rmlB, 175
rmlD). Gene for biosynthesis of glycerol-1-phosphate (gct) were present in both 16F and 16A with 176
77% identities in BLASTp alignment (21). The presences of these similar genes indicated that 16F 177
and 16A CPS both contain glucose, rhamnose and glycerol-1-phosphate in their repeat unit 178
structures. However, genes for synthesis of galactofuranose(glf) presented in serotype 16A but not 179
in 16F. Moreover, they have significantly different glycosyltransferase (GTs) genes and 180
oligosaccharide repeat unit polymerases (Wzy). The Wzy27 in 16F and Wyz15 in 16A showed only 181
28% identities in BLASTp comparison. According to 16F and 16A CPS structures, Wzy27 is 182
assigned to polymerize at β-D-Glcp-(1-3)-α-L-Rhap, while Wzy15 is assigned to polymerize at β-D-183
Glcp-(1-3)-β-D-Galf. 184
The cps locus of 16F contained three GT genes. The GT (WchF) has been shown to be encoding the 185
β-1-4 rhamnosyltransferase for addition of the second sugar(22). The GT gene (wciU) in 16F cps 186
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locus, also present in serogroup 18, which added α-D-Glcp or α-D-GlcNAc to the 3-position of β-L-187
Rhap(19). Therefore, the only unassigned GT (WcxN) should be responsible for addition of α-L-188
Rhap to the 3-position of α-D-Glcp, based on the 16F CPS structure we determined in this paper. 189
The putative glycerol phosphotransferase(WcxP) and putative LicD-family 190
phosphotransferase(WcxQ) were responsible for addition of Gro-1-P substitution to the 4-position 191
and 6-position of α-D-Glcp and α-L-Rhap, respectively, but it was hard to distinguish which specific 192
linkage catalyzed either one of them was responsible for. The WcxM, a putative acetyl transferase, 193
catalyzed O-acetylation at the 2-position of β-L-Rhap, which was consistent with serotype 194
18F(1)(23). 195
Serotype 16A cps locus had five GT genes. The GT (WchK) that is found in 13 different serotypes, 196
was assigned as β-1-4-galactosyltransferase and WchJ was suggested to enhance activity of 197
WchK(18)(24). The WcyK, also found in serogroup 11(25), catalyzed linkage of α-D-Galp-(1-3)-β-198
D-Galp. The WcxS catalyzed the linkage of α-L-Rhap-(1-3)-α-D-Galp, which were shown for 199
serotype 45(19). The WciB, present in 25 pneumococcal serotypes(Table 4), was assigned as β-1-3-200
galactofuronosyl transferase (26)(27)(Table 4). Thus, the only unassigned GT(WcxT), by process of 201
elimination, was assigned to be responsible for catalyzing the linkage of α-L-Rhap-(1-2)-α-L-Rhap. 202
The WciG, a putative acetyl transferase, catalyzed 2-O-acetylation at β-D-Galf(28)(29)(27) (30). 203
The WcxR is putative LicD-family phosphotransferase which was assigned to catalyze 204
phosphorylation at the 6-position of α-D-Galp. The analysis of the biosynthesis of 16F and 16A CPS 205
was in an agreement with determined CPS repeat unit structure and functions of related genes 206
reported in the literature. 207
When compared to other serogroups, 16F cps locus showed high similarity to serotype 28A and 28F 208
and it was in the same subcluster of cluster 2 (18)(23)(Fig. 4). Not only containing the same 209
conserved regulatory genes, they have highly similar WchA, GTs, acetyl transferase (WcxM) and 210
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flippase (Wzx). Main differences in 28F and 28A cps loci were glycerol-2-phosphate biosynthetic 211
genes (gtp1, gtp2, gtp3), compared to glycerol-1-phosphate (gct) in 16F (Fig.4). Thus, the repeat 212
unit structure of 16F CPS should have similarities to 28F and 28A CPS structure, which are 213
unfortunately both unknown. 214
Serological cross-reactions of diagnostic antisera between serogroup 16 and other serotypes 215
When serotyping S. pneumoniae, serogroup 16 reacts with pool antiserum D and was identified by 216
group antisera 16. Furthermore, factor antisera 16b and 16c was used to determine serotype 16F and 217
16A (Table 5). Positive Quellung reactions were observed between serotype 16F and 16A and 218
factor antiserum 11c. Factor antiserum 11c reacted with serotype 11A, 11C and 11D and is reported 219
to bind to P-Gro and OAc groups (25). Moreover, the cross-reaction between serotype 16F and 220
factor serum 11d was previously observed (18). These cross-reactions indicated similar antigenic 221
properties among them. However, the cps gene locus of serotypes 11A do not show high similarity 222
to 16F cps locus apart from regulatory genes, wchA and gct(Fig.4), while 16A shared some similar 223
GTs (WchJ, WchK and WcyK) with serogroup 11. The common parts of the 16F CPS repeat unit 224
structure compared with that of 11A, are that they consist of a linear tetrasaccharide backbone with 225
glycerol-1-phosphate branches and O-acetyl substitution. The O-acetylation was reported to be 226
crucial to the antigenicity of serogroup 11(25)(31). The cross-reactions between group 16 and factor 227
antiserum 11c confirmed that factor 11c epitope was associated with P-1-Gro and OAc groups. 228
Serotype 16F cross-reacted with factor antiserum 28b, which was used for serotyping 28F. Thus, it 229
can be predicted that the CPS structure of 28F might be similar to 16F, as they have highly similar 230
cps genes and common serological activity. No cross-reaction was observed between factor 231
antiserum 11c and serotype 28F, which has genes to express P-2-Gro synthase, indicating that factor 232
antiserum 11c might react specifically to P-1-Gro and not P-2-Gro. Additionally, serotype 28F and 233
28A reacted with factor antiserum 23d, which is used for serotyping 23B. CPS structures of group 234
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23 all contain P-2-Gro(32). However, Factor antiserum 23d only reacts with serotype 23B of 235
serogroup 23. Thus, it suggests that there might be other similar structural properties other than P-2-236
Gro between serogroup 28 and serotype 23B. However, these assumptions about serogroup 28 need 237
further structural information to be confirmed. 238
Summary of results 239
The CPS repeat unit structures of serotype 16F and 16A were elucidated using NMR spectroscopy 240
and chemical analysis (Fig. 2), which provide first comprehensive understanding of serogroup 16 241
CPS. Furthermore, based on the determined CPS structures, a comparison analysis of CPS 242
biosynthesis of serotype 16F and 16A with related serotypes that share same biosynthetic genes 243
revealed functions of previously unknown glycosyltransferases WcxN and WcxT (Table 4). Finally, 244
the serogroup 16 CPS were subjected to serological studies to determine any relevant cross-245
reactions with antisera from the genetically similar serogroup 28, where it was shown that the CPS 246
of serotype 16F and 28F have similar antigenic properties, as they both react with factor serum 28b 247
which is used for 28F serotyping. Thus, it could be predicted that the CPS structure of serotype 28F 248
should be similar to serotype 16F, since they have highly similar cps genes and serological 249
behavior. 250
MATERIALS AND METHODS 251
Sample preparation 252
Purified pneumococcal capsular polysaccharide (CPS) from serotypes 16A and 16F, as well as 253
rabbit antisera, were produced by SSI Diagnostica, Hillerod, Denmark. The polysaccharide samples 254
were dissolved in D2O (99.9 %, Sigma) to concentrations of approximately 1.5% w/v (10 mg in 600 255
μL). 256
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Ultra sonicated 16F CPS were obtained by sonication of the CPS (10mg in 5 mL 2M KCl) using a 257
LABSONIC® P ultrasonic homogenizer with a 3 mm Probe for 2 hours under an ice bath, 258
following this it was desalted and concentrated using centrifugal filters (Amicon® Ultra-4, 3 kDa). 259
De-OAc and de-P-Gro were performed using methods described by Richards and Perry(33). De-260
OAc removed only acetyl groups, while de-P-Gro removed both acetyl and glycerophosphate 261
groups. De-OAc CPS were obtained by treating approximately 5-10 mg of each polysaccharide with 262
1 mL of a 0.2 M NaOH in D2O solution at room temperature for 2 h, which was subsequently 263
neutralized with 2M HCl, followed by desalting and removal of residuals using centrifugal filters 264
(Amicon® Ultra-4, 3 kDa). Dephosphorylated CPS were generated by treating approximately 10 265
mg of each CPS in 2 mL of 1 M NaOH, containing a trace amount of sodium borohydride, at 100 266
°C for 4 h in screw-capped glass tubes filled with nitrogen. Then the solutions were cooled in an ice 267
bath and neutralized with acetic acid, followed by desalting and removal of residuals using 268
centrifugal filters (Amicon® Ultra-4, 10 kDa). Finally, these samples were transferred to 5 or 3 mm 269
NMR tubes. 270
NMR spectroscopy 271
NMR spectroscopy were used to determine repeat unit structures of serotypes 16A and 16F CPS. 272
The NMR experiments were carried out on an a Bruker Avance III (799.90 MHz for 1H and 201.14 273
MHz for 13C) equipped with a 5 mm TCI 1H/(13C, 15N) cryoprobe or a Bruker AVANCE 600 MHz 274
instrument with 5 mm SmartProbe BB(F)-H-D(15N-31P, 1H, 19F). 275
All spectra of 16A CPS were recorded at 313 K. While 16F CPS spectra were recorded at 333K or 276
323K. The assignments of 16A and 16F CPS repeat units were performed using the following 277
experiments: 1D 1H and 31P and the 2D NMR spectra, including DQF-COSY, TOCSY with 80 ms 278
mixing time, NOESY with 200-400 ms mixing time, HSQC, HMBC optimized for 8 Hz long range 279
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coupling constants, 1H–13C HSQC-TOCSY with 80 ms mixing time, CLIP-HSQC and 1H–31P 280
HMBC. Experiments were conducted with standard gradient enhanced Bruker pulse sequences, 281
except CLIP-HSQC(11). 282
NMR spectra were processed with Topspin 3.5 (Bruker) using extensive zero filling in all 283
dimensions. The one-dimensional 1H spectra were processed with an exponential window function 284
with a line broadening of 0.3 Hz for 1H spectra. All 2D spectra were processed with shifted sine bell 285
window functions in both dimensions. All spectra were referenced to residual cell wall 286
polysaccharide phosphocholine signals (1H 3.2 ppm and 13C 54.5 ppm and the shielded 31P signal at 287
1.30 ppm)(13). 288
General chemical analysis 289
The monosaccharide composition was determined by GC after hydrolysis (34) (2 M TFA at 120 290
°C, 2 h), addition of xylose as internal standard, reduction (NaBH4, 16 h in the dark) and 291
peracetylation (acetic anhydride and pyridine (1:1) 85 °C, 10 min, twice). GC analysis was 292
performed on a HP 5890 (series II) gas chromatograph with a flame-ionization detector and a 293
column (30 m × 0.25 mm × 0.25 μm film thickness, Agilent) of 5% phenylmethylsiloxane (HP-294
5MS); helium was used as carrier gas (70 kPa). The temperature in GLC was 150°C for 3 min, and 295
then increased with 3°C/min to 320°C. A mixture of standard monosaccharides was used for sugar 296
identification and xylose for quantification. 297
Methylation was performed using Ciucanu-Kerek's method (35). Prior this the removal of 298
phosphate from CPS samples was done by using 48% HF for 48 h at 4 °C, followed by removal of 299
HF by evaporation under N2 while suspended in DMSO for 16h at 60 °C, and then methylated with 300
iodomethane in the presence of NaOH. The product was hydrolyzed with 4 M TFA for 4 h at 301
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100 °C, reduced with NaBD4 and peracetylated with acetic anhydride in pyridine. Partially 302
methylated acetylated alditols were analyzed by GC-MS. 303
The content of total phosphate was determined by colorimetric analysis according to Lowry et 304
al(36). The samples were subjected to digestion reagent (30.6 mL conc. H2SO4, 6.7 mL 70% 305
HClO4 and 62.7 mL H20) at 100 °C for 1h and then at 165 °C for 2h. Subsequently, it was cooled 306
down to room temperature and freshly prepared color reagent was added (1 mL 1 M sodium acetate, 307
1 mL 2.5% ammonium molybdate solution, 7 mL H2O, 1 mL freshly prepared ascorbic acid; 37 °C 308
for 90 min). The extinction of the ammonium phosphomolybdate complex was measured at λ = 820 309
nm (HELIOS BETA 9423 UVB 1002E spectrophotometer, Thermo Electron Ltd., Altrincham, 310
Cheshire, UK). For quantification, 5 mM Na2HPO4 solution was used as external standard. 311
The absolute configuration of Rha and Glc was determined by GLC (37), by comparison with 312
authentic standards of the acetylated O-[(R)-2-Octyl] glycoside after methanolysis (0.5 M 313
HCl/MeOH, 85 °C, 3.5 h), butanolysis (2 M HCl/(R)-2-Octanol, 65 °C, 4 h) and peracetylation (85 314
°C, 10 min). The absolute configuration of Gal was determined by GLC, by comparison with 315
authentic standards, of the acetylated O-[(S)-2-butyl] glycoside after methanolysis (2M HCl/MeOH, 316
85 °C, 45min), butanolysis (2 M HCl/(S)-2-Butanol, 65 °C, 4 h) and peracetylation(85 °C, 10 min). 317
Genetic analysis of serogroup 16 318
The published cps loci sequences (accession number of serotype 16F, 16A, 28F, 28A, 11A: 319
CR931668.1, CR931667.1, CR931693.2, CR931692.1, CR931653.1, respectively) have been 320
downloaded from GenBank (https://www.ncbi.nlm.nih.gov/nuccore). Pairwise protein sequence 321
identity has been assessed using BLASTp. Comparison of similarities of the gene products 322
(TBLASTX) of the cps biosynthetic loci was performed by the Artemis comparison tool (ACT)(38). 323
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The Quellung reaction was performed at SSI Diagnostica using factor antisera (SSI Diagnostica 324
A/S, Hilleroed, Denmark). For the Quellung reaction, bacteria were grown in horse blood agar plate 325
(SSI Diagnostica A/S, Hilleroed, Denmark) overnight. 2-4μL of culture was added onto a glass 326
slide and mixed with the same amount of factor antisera. The mixture was then observed for capsule 327
swelling reaction in a phase contrast microscope. 328
ACKNOWLEDGEMENTS 329
The 800 MHz NMR spectra were recorded on the NMR Center-DTU, supported by the VIllum 330
Fondation. C.L. appreciate the financial support of China Scholarship Council (PhD scholarship 331
No. 201708310119) and Oticon Fonden for financial support during her external stay in Research 332
Center Borstel. We thank Kasper Enemark-Rasmussen for excellent technical assistance with NMR 333
instrument setup, Katharina Jakob (Research Center Borstel) for general chemical analysis and 334
Katrine Helander Pedersen for serological test. 335
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447
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Table 1 Results of monosaccharides compositional analysis, phosphate, methylation and absolute 448
configuration analysis 449
Serotype Rha (nmol/mg)
Glc (nmol/mg)
Gal (nmol/mg)
PO4 (nmol/mg) Linkage type L/ D-
configuration
16F CPS 1545 1276 - 1770 2-linked Rha, 4-linked Glc,
3-linked Glc L-Rha, D-Glc
Ratio 1.2 1.0 - 1.4
16A CPS 1455 686 2005 942 1,2-linked Rha, 1,3-linked Rha, 4-linked Glc, 3-linked Glc, 1,3-linked-Galf, 1,2,3-
linked Gal
L-Rha, D-Glc, D-Gal Ratio 2.1 1.0 2.9 1.4
Table 2 1H and 13C NMR chemical shifts (δ, ppm) assignment of sonicated 16F CPS, de-OAc 16F 450
CPS and de-P-Gro 16F CPS repeat unit as well as important HMBC/NOE correlations 451
Sample Residue 1 2 3 4 5 6 HMBC/ NOE
Sonicated 16F CPS
3)-α-Rhap-(1- A
5.107 4.268 3.964 4.110 4.128 1.286 A1-C3 100.69 70.56 80.99 75.72 68.33 17.34
3)-β-Rhap-(1- B
5.037 5.634 3.866 3.504 3.506 1.350 B1-D4 99.63 68.92 75.25 71.15 72.67 17.22
OAc : 2.154 / 20.74 C=O: 174.07 B2
3)-α-Glcp-(1- C
5.000 3.657 3.788 3.543 3.985 4.058 C1-B3 95.18 71.91 79.94 67.98 71.15 64.70
4)-β-Glcp-(1- D
4.536 3.373 3.580 3.654 3.447 3.755/3.860 D1-A3 104.56 73.61 75.48 77.68 74.66 61.13
P1-1-Gro 3.921/ 3.855 3.874 3.666/3.646
/3.588
66.92 71.26 62.70 P1 0.6031 A4
P2-1-Gro 3.874 3.874 3.666/3.646
/3.588
67.04 71.26 62.70 P2 1.2222 C6
de-OAc 16F
3)-α-Rhap-(1- A
5.116 4.320 4.010 4.150 4.164 1.292 A1-C3 100.81 70.44 80.88 76.19 68.33 17.22
3)-β--Rhap-(1- B
4.850 4.269 3.670 3.473 3.417 1.324 B1-D4 100.92 67.75 78.30 70.79 72.67 17.22
3)-α-Glcp-(1- C
5.063 3.700 3.869 3.575 4.088 4.117/4.094 C1-B3 96.00 72.20 80.06 67.98 71.26 64.93
4)-β-Glcp-(1- D
4.583 3.397 3.653 3.653 3.508 3.836/3.887 D1-A3 104.56 73.73 75.60 77.24 74.90 61.30
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P1-1-Gro 3.959/ 3.910 3.889 3.666/3.598
67.28 71.15 62.71 P1 0.2338 A4
P2-1-Gro 3.924 3.889 3.647/3.590 67.39 71.15 62.71
P2 0.8944 C6
de-P-Gro 16F
3)-α-Rhap-(1- A
5.104 4.281 3.920 3.604 4.064 1.251 A1-C3 101.16 70.44 80.64 71.38 69.15 16.99
3)-β--Rhap-(1- B
4.850 4.275 3.659 3.465 3.411 1.32 B1-D4 100.92 67.63 78.06 70.68 72.67 17.23
3)-α-Glcp-(1- C
5.067 3.662 3.850 3.508 3.955 3.772/3.790 C1-B3 95.88 72.2 80.17 68.33 72.32 60.78
4)-β-Glcp-(1- D
4.659 3.374 3.637 3.637 3.508 3.811/3.903 D1-A3 104.09 73.84 75.84 77.12 74.90 61.18
Table 3 1H and 13C NMR chemical shifts (δ, ppm) assignment of native 16A CPS, de-OAc 16A and 452
de-P-Gro CPS repeat unit as well as important HMBC/NOE correlations 453
Sample Residue 1 2 3 4 5 6 HMBC/ NOE
Native 16A CPS
3)-β-Galf-2-OAc-(1- A 70%
5.395 5.155 4.407 4.250 3.966 3.706/3.674 A1-D3
107.55 82.45 83.27 83.27 70.6 63.27
OAc: 2.119/20.75 C=O: 173.45 A2
3)-β-Galf-(1- A’ 30%
5.2406 4.345 4.267 4.191 3.952 3.706/3.674 A'1-D'3 109.66 80.45 85.14 82.45 70.83 63.27
2)-α-Rhap-(1- B
5.192 4.057 3.949 3.489 3.805 1.29 B1-C3 100.98 78.46 70.48 72.71 69.78 17.23
3)-α-Galp-(1- C
5.131 3.981 3.993 4.113 4.328 3.960 C1-F3 96.29 68.37 76.70 68.96 70.01 64.44
3)-α-Rhap-(1- D
4.947 4.164 3.895 3.533 3.753 1.254 D1-B2 102.27 70.48 77.87 71.54 69.78 17.23
D’3 3.864 /78.11
4)-β-Glcp-(1- E
4.673 3.340 3.668 3.665 3.58 3.962/3.830 102.15 73.18 74.70 79.04 75.29 60.45 E1-A3
E' 4.637/102.39
3)-β-Galp-(1- F
4.524 3.653 3.771 4.147 3.714 3.744/3.775 103.33 70.01 78.22 65.56 75.53 61.39 F1-E4
Gro-1-P 3.901/3.845 3.881 3.647/3.590
66.79 71.19 62.57
P 0.9144 C6
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de-OAc 16A CPS
3)-β-Galf-(1- A
5.230 4.342 4.264 4.178 3.943 3.708/3.656 A1-D3 109.79 80.54 85.20 82.46 70.73 63.34
2)-α-Rhap-(1- B
5.191 4.057 3.943 3.493 3.802 1.287 B1-C3 101.11 78.61 70.73 72.66 69.93 17.21
3)-α-Galp-(1- C
5.124 3.967 3.998 4.111 4.334 3.951 C1-F3 96.29 68.32 76.84 68.96 70.09 64.46
3)-α-Rhap-(1- D
4.941 4.170 3.857 3.513 3.752 1.252 D1-B2 102.39 70.57 78.13 71.54 69.77 17.21
4)-β-Glcp-(1- E
4.639 3.329 3.665 3.669 3.611 3.963 E1-A3 102.39 73.14 74.75 78.93 75.39 60.43
3)-β-Galp-(1- F
4.526 3.642 3.767 4.147 3.712 3.744/3.775 F1-E4 103.36 70.09 78.13 65.59 75.55 61.41
Gro-1-P 3.650/3.583 3.877 3.896/3.842
62.54 71.21 66.88 P 0.7042
de-P-Gro 16A CPS
3)-β-Galf-(1- A
5.230 4.342 4.269 4.178 3.936 3.702/3.662 A1-D3 109.79 80.47 85.05 82.41 70.86 63.29
2)-α-Rhap-(1- B
5.184 4.059 3.936 3.494 3.790 1.281 B1-C3 101.05 78.42 70.39 72.62 69.80 17.15
3)-α-Galp-(1- C
5.137 3.976 3.973 4.059 4.188 3.704 C1-F3 95.89 68.34 77.01 69.39 71.33 61.24
3)-α-Rhap-(1- D
4.941 4.169 3.851 3.513 3.748 1.251 D1-B2 102.34 70.45 78.13 71.50 69.80 17.26
4)-β-Glcp-(1- E
4.643 3.330 3.665 3.660 3.614 3.807/3.797 E1-A3 102.28 73.14 74.67 78.89 75.31 60.48
3)-β-Galp-(1- F
4.504 3.662 3.775 4.161 3.699 3.780/3.733 F1-E4 103.34 69.98 77.66 65.29 75.55 61.47
Table 4 Serotype 16F and 16A CPS biosynthetic genes, their predicted functions or products, 454
structures they catalysed and related serotypes containing same genes. 455
Genea No. of
occurence of gene
Product/Fuction
Structure Serotypes containing the gene
donor linkage acceptor CPS Structure known
CPS Structure unknown
wchF 27 GT L-Rhap β 1-4 β-D-Glcp 2,7F,7A,7B,17F,18F,18A,
18B,18C,22F,23F,27,32F,32A,16F
wciU 7 GT D-GlcpNAc α 1-3 β-L-Rhap 18A
28F,28A D-Glcp α 1-3 β-L-Rhap 18F,18B,18C,16F
wcxN 3 GT L-Rhap α 1-3 α-D-Glcp 16F 28F,28A
wcxM 4 acetyl transferase β-L-Rhap-2-OAc 18F,16F 28F,28A
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wzy27b 1 polymerase β-D-Glcp-(1,3)-α-L-Rhap 16F
gct 13 glycerol-1-phosphate biosynthetic protein
P-1-Gro 11A,11C,11D,11E,18F, 18A,18B,18C,45,16F,16A
Rib-ol-1-P 11B,11F(truncated gct)
wcxP 3 sugar/polyalcohol phosphate transferase
D-Glcp-6-P-1-Gro / L-Rhap-4-P-1-Groc 16F 28F,28A
wcxQ 3 sugar/polyalcohol phosphate transferase
D-Glcp-6-P-1-Gro / L-Rhap-4-P-1-Groc 16F 28F,28A
wchK 13 GT D-Galp β1-4 β-D-Glcp 11F,11A,11B,11C,11D, 11E,13,14,15F,15A,15B,
15C,16A
wchJ 13 GT enhancer
wcyK 7 GT D-Galp α 1-3 β-D-Galp 11F,11A,11B,11C,11D, 11E,16A
wcxS 2 GT L-Rhap α 1-3 α-D-Galp 45,16A
wcxT 1 hypothetical protein L-Rhap α 1-2 α-L-Rhap 16A
wciB 25 GT D-Galf β 1-3
β-D-Galp 10F,10A,10B,10C,29,39,47F,47A
43
β-D-Glcp 17A,20A,20B,33F,33A,35F, 35A,35B,35C,41F,41A,42
α-D-Glcp 34
α-L-Rhap 16A
37(synthase pathway)
wzy15b Polymerase β-D-Glcp-(1,3)-β-D-Galf 16A
wciG 16 acetyl transferase
β-D-Galf-2-OAc 20A,20B,33F,33A,33B,33D,35F,35A,35B,47F,16A
β-D-Glcp-2,3-OAc 13
β-D-Galf-5,6-OAc 42(containing wciG and wciE)
NO OAc 35C(containing wciG and wciE), 10F,10C
wcxR 1 sugar/polyalcohol phosphate transferase Gro 1-P-4 α-D-Galp 16A
a Regulatory genes (wzg, wzh, wzd and wze), initial transferase (WchA), L-rhamnose and 456
galactofuranose synthetic genes are not included. 457
b The wzy27 and wzy15 mean that they were belong to Wzy homology groups 27 and 15, 458
respectively. 459
c The specific functions of two sugar/polyalcohol phosphate transferases (WcxP and WcxQ) can not 460
be distinguished. 461
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Table 5 Cross-reactions between serogroup 16 and serogroup 28 using Quellung reaction 462
serotype factor sera 16b 16c 28b 28c 11b 11c 11f 11g 23b 23c 23d
16F +++ - +++ - - +++ - - - - - 16A - +++ - - - +++ - - - - - 28F - - +++ - - - - - - - +++ 28A - - - +++ - - - - - - +++
463
Fig. 1 Expansion of 1H spectra of native16F CPS, Ultra sonicated16F CPS, de-OAc 16F CPS and 464
de-P-Gro 16F CPS 465
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466
Fig. 2 The repeat units structures of 16F and 16A CPS 467
468
Fig. 3 Expansion of 1H and HSQC spectrum of native 16A CPS(313K) and de-OAc 16A 469
CPS(298k) 470
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471
Fig. 4 Comparison of serogroup 16 CPS biosynthetic gene loci and repeat unit structures with other 472 serotypes. (A) cps gene loci of S. pneumonia serogroup 16 and related serotypes; Red frames 473 highlight genes highly similar to 16F cps gene locus; (B) CPS repeat unit structures and 474 biosynthetic enzymes(red characters) that are responsible for corresponding structures indicted by 475 blue arrow; 476
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